Imbalanced Expression of Th2 and Treg Cell-related Parameters in Peripheral Blood Mononuclear Cells in Patients with Allergic Asthma.

Background
The imbalance between Th2 and Treg cells plays fundamental role in the pathogenesis of allergic asthma. The current study aimed at assessing the expression of some Th2 and Treg cell-related parameters in patients with allergic asthma.


Material and Methods
The serum and peripheral blood mononuclear cell (PBMC) samples were collected from 30 patients with asthma and 36 healthy subjects. The serum levels of transforming growth factor (TGF)-β, interleukin (IL)-4, as well as the expression levels of GATA3 and FOXP3 genes in PBMCs were determined by the enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR), respectively. The PBMCs were cultured for 48 hours with/without phytohemagglutinin (PHA) stimulation. The TGF-β and IL-4 levels in supernatants were also determined.


Results
The serum levels of IL-4, the expression level of GATA3, and GATA3/FOXP3 ratio in patients with asthma were significantly higher than healthy subjects (P <0.002, P <0.001, and P <0.004, respectively). The FOXP3 expression did no differ between the two groups. The serum level of TGF-β as well as its secretion profile in non-stimulated and stimulated PBMCs isolated from patients with asthma were significantly higher than those of the controls (P <0.03, P <0.001, and P <0.001, respectively). The serum TGF-β levels in severe asthma were significantly higher than moderate asthma; whereas the TGF-β secretion by PHA-stimulated PBMCs isolated from moderate asthma was higher than that of severe pattern of the disease (P <0.001 and P <0.05, respectively). The GTAT3/FOXP3 expression ratio in moderate asthma was significantly higher than severe form (P <0.04).


Conclusion
The results confirmed a Th2 cell-biased pattern and possible contribution of TGF-β in allergic asthma. TGF-β may have different expression patterns in moderate and severe asthma and the two forms of the disease may have differences in some main immunological parameters.

The Treg cells constitute 5% to 15% of the total circulating CD4 + T lymphocytes divided into two subsets.
The natural Tregs (nTreg) cells are generated in the thymus, whereas the induced Treg (iTreg) cells are differentiated from peripheral CD4 + T lymphocytes after antigenic recognition and the existence of IL-2 and TGF-β (13,14). The immunosuppressive effects of Treg cells are exerted by releasing TGF-β, IL-10, and IL-35 that are necessary to establish tolerance to self-constituents or foreign antigens (13,15). The FOXP3 protein is considered as a master transcriptional factor of Treg cells and its gene is mapped to the X chromosome (15,16). Genetic abnormalities in FOXP3 gene result in the inflammatory   syndrome  named  immune  dysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) (17).
There is evidence indicating that Th2/Treg imbalance plays an essential role in the development of allergic sicknesses (18). The results of a number studies indicated that the count of Th2 cells was enhanced, while the count of Treg cells was diminished in the peripheral blood of patients with asthma or in the models of experimentallyinduced allergic asthma, representing the contribution of the Th2/Treg imbalance in the pathogenesis of allergic asthma (7,19).
It should be noted that the measurement of transcription factors GATA3 and FOXP3 may more reliably indicate the Th2/Treg cell development than the single assessment of a Th2 or Treg cell-related element. Therefore, it seems that the measurement of the transcriptional factor level is more important to evaluate the balance in Th2/Treg immune responses.
There are some controversies regarding the association of Th2 and Treg cell-related parameters with asthma severity. For example, the results of a number of studies demonstrated decreased serum level of TGF-β in patients with asthma (19,20), while others reported elevated levels of this cytokine in the patients (21)(22)(23). Moreover, the exact association of Th2 and Treg cell-related parameters with asthma severity is still unclear.
The current study aimed at determining the key parameters of Th2 (IL-4 and GATA3) and Treg (TGF-β and

The serum and PBMC supernatant levels of TGF-β and IL-4
The serum and PBMC supernatant levels of TGF-β and IL-4 were quantified using commercial ELISA (the enzyme-linked immunosorbent assay) kits for human TGFβ and IL-4 (eBioscience, USA).

Serum IgE concentrations
Serum total IgE concentrations (IU/mL) were measured by the commercial ELISA kit (Monobind Inc., Costa Mesa, CA, USA).

Statistical analysis
Data were expressed as mean ± standard error of the mean (SEM). Differences in variables were analyzed by appropriate statistical tests including ANOVA, the Student t, Kruskal-Wallis, and Mann-Whitney U tests; P values <0.05 were regarded statistically significant.

Serum levels of TGF-β and IL-4 in patients with asthma and healthy controls
The serum levels of TGF-β and IL-4 were significantly higher in patients with asthma than the healthy controls The serum TGF-β levels of asthmatic were significantly higher than in healthy subjects. The serum IL-4 levels in patients with asthma were significantly higher than in healthy subjects. The serum TGF-β levels in patients with severe asthma were significantly higher than in patients with moderate asthma and healthy subjects.    Figure 5).
The expression of FOXP3 in PBMCs isolated from patients with moderate asthma (2.33±0.90) was higher than that of the ones with severe asthma (1.12±0.33) as well as the healthy subjects, but the differences were not statistically significant ( Figure 6). The GTAT3 expression in both patient groups with moderate-and severe asthma were significantly higher than healthy control group. The FOXP3 expression in patients with moderate asthma was higher than those with severe asthma and also healthy subjects, but the differences were not significant.   Table 2. In both groups of healthy subjects and patients with asthma, TGF-β and IL-4 production by PHA-stimulated PBMCs were significantly higher than those of the unstimulated cells (P <0.05 and P <0.001, respectively) ( Figures 8 and 9). In both healthy-and asthmatic groups, the PHA-induced TGF-β secretion were significantly higher than nonstimulated culture. The levels of TGF-β production by non-stimulated and PHA-stimulated PBMCs from patients with asthma were significantly higher than in the same cultures from healthy subjects. In both healthy-and asthmatic groups, the PHA-induced IL-4 secretion were significantly higher than nonstimulated culture. The levels of IL-4 production by non-stimulated PBMCs from patients with asthma were significantly higher than in the same cultures fromhealthy subjects. The levels of TGF-β and IL-4 production by nonstimulated PBMCs in patients with asthma were significantly higher than those of healthy controls (P <0.001 and P <0.03, respectively) (figures 8 and 9). Moreover, the production of PHA-stimulated TGF-β by PBMCs isolated from patients with asthma was significantly higher than that of healthy controls (P <0.001) (Figure 8). There was no significant difference in terms of the production of PHAstimulated IL-4 between patients and healthy individuals, although the parameter was higher in patients groups (P=0.34) (Figure 9).
The production of TGF-β by unstimulated and PHAstimulated PBMCs isolated from patients with moderate asthma were higher than those of the ones with severe asthma, although the difference was significantly higher in PHA-stimulated cells (P <0.05) ( Table 2). No significant difference was observed between patients with moderate and severe asthma regarding the production of IL-4 by non-stimulated and PHA-stimulated PBMCs ( Table 2).
There was no significant difference between males and females in patients or in the control groups regarding the production of TGF-β and IL-4 by unstimulated and PHAstimulated PBMCs (Table 3).

DISCUSSION
The results of the current study indicated that the The results of the current study also showed that the serum levels of TGF-β in patients with asthma were significantly higher than those of the healthy individuals.
There were some controversies regarding the TGF-β levels The results of the current study also indicated that GATA3/FOXP3 expression ratio in patients with asthma was significantly higher than those of healthy individuals.
Since The results of the current study also indicated that the serum levels of TGF-β in patients with severe asthma were higher than those of the ones with moderate asthma.
However, in controversy to serum levels of TGF-β, the production of this cytokine by PHA-stimulated PBMCs isolated from patients with moderate asthma was higher than that of the ones with severe asthma. These differences may be attributed to different in vivo cell producers of TGF-β. As mentioned above, TGF-β is produced by some promote the development of severe allergic asthma due to their link to airway neutrophilia (37). Therefore, TGF-β may also influence the severity of allergic asthma.
Accordingly, targeting TGF-β or its related signaling pathways may lessen asthma severity.
However, the current study results demonstrated that GTAT3/FOXP3 expression ratio in patients with moderate asthma was significantly higher than that of the subjects with severe asthma. Therefore, it seems that moderate and severe asthma may have differences in some important immunological aspects. Other pathological mechanisms of Th2-dependent inflammation (such as Th17, Th9, and Th22, or even Th1 immune responses) may also contribute to the pathogenesis of severe asthma. Indeed, high levels of Th1 cytokines are found in humans and mice with severe asthma (38). Therefore, a wide range of immunopathological responses may involve in the development of severe asthma. Accordingly, a combination of therapies is needed to control asthma severity and prevent its exacerbations.
In the current study, one Th2 cell-associated cytokine The improvement of Th2/Treg immune responses should be considered more to design effective therapeutic strategies to treat allergic asthma.
Elevated serum levels of TGF-β and increased production of TGF-β by unstimulated and PHA-stimulated PBMCs isolated from patients with asthma indicate that TGF-β may contribute to immunopathogenesis of allergic asthma. Therefore, targeting TGF-β or its related signaling pathways may have therapeutic benefits for allergic asthma.
The serum level of TGF-β in patients with severe asthma was higher than that of the ones with moderate asthma, whereas the TGF-β production by PHA-stimulated PBMCs isolated from patients with moderate asthma was also higher than that of the patients with severe asthma.
The current study results showed that TGF-β may have different effects on moderate and severe asthma.
GTAT3/FOXP3 expression ratio in patients with moderate asthma was significantly higher than that of the ones with severe asthma. Therefore, moderate and severe asthma may have significant differences in some main immunological parameters.